Comparative Evaluation of Real-Time PCR Assays for Detection of the Human Metapneumovirus
Infectious Diseases Research Center, Centre Hospitalier Universitaire de Québec (CHUQ-CHUL),1 Department of Medical Biology, Laval University, Québec City, Canada2:, http://www.100md.com
Received 16 January 2003/ Returned for modification 13 March 2003/ Accepted 5 May 2003:, http://www.100md.com
ABSTRACT:, http://www.100md.com
The human metapneumovirus (hMPV) is a new member of the Paramyxoviridae family associated with acute respiratory tract infections in humans. The objective of this study was to compare the sensitivity of real-time RT-PCR assays performed in a LightCycler instrument and designed to amplify the viral nucleoprotein (N), matrix (M), fusion (F), phosphoprotein (P), and polymerase (L) genes. In a first evaluation of 20 viral cultures with characteristics compatible with hMPV cytopathic effect, the PCR positivity rates were 100, 90, 75, 60, and 55% using primers for the N, L, M, P, and F genes. In a second evaluation of 10 nasopharyngeal aspirates from children with bronchiolitis and found to be positive for the hMPV N gene, the PCR positivity rates for the L, M, P, and F genes were 90, 60, 30, and 80%, respectively. The analytic sensitivity of the real-time RT-PCR assay for the hMPV N gene was 100 copies using a transcribed viral plasmid. In conclusion, real-time PCR assays aimed at amplifying the N and L genes which are coding for two internal viral proteins appear particularly suitable for hMPV diagnostic.
INTRODUCTION9m11-v2, 百拇医药
The human metapneumovirus (hMPV) is a newly identified negative-sense RNA virus that has been preliminary assigned to the Metapneumovirus genus within the Paramyxoviridae family (13, 14). This virus differs from the human respiratory syncytial virus (hRSV), another paramyxovirus belonging to the Pneumovirus genus, by virtue of a lack of two nonstructural proteins (NS1 and NS2) and by a different genomic organization for the other common genes, i.e., the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), polymerase (L), attachment glycoprotein (G), M2, and small hydrophobic protein (SH).9m11-v2, 百拇医药
HMPV has been shown to replicate slowly in a few cell lines including tertiary monkey kidney (TMK) cells with cytopathic effects consisting mainly of syncytium formation appearing more than 10 to 14 days postinoculation (2, 14). Virus-infected TMK cell-culture supernatants also do not display any hemagglutinating activity. Molecular detection of hMPV in clinical samples has been mainly achieved by amplifying the F, N, or L genes although no systematic evaluation of such different PCR assays has been performed (2, 5, 12, 14; M. D. Nissen, D. J. Siebert, I. M. Mackay, T. P. Sloots, and S. J. Withers, Letter, Med. J. Aust. 176:188, 2002). A rapid and sensitive PCR assay for hMPV is required in view of its relatively inefficient growth using conventional cell culture methods and its role as a respiratory pathogen in young children, elderly subjects, and immunocompromised patients (2, 4, 9, 14; Nissen et al., letter). In this study, we sought to compare various real-time PCR assays for optimal detection of hMPV in both infected cell cultures and respiratory clinical specimens.
MATERIALS AND METHODS#}^-g2], 百拇医药
Viral isolates and clinical samples. A subset of 20 viral isolates were selected from respiratory samples collected between 2000 and 2002 on the basis of the following characteristics: (i) slow growth on LLC-MK2 cells (a continuous cell line from rhesus monkey kidneys) with no growth on Madin-Darby canine kidney, Vero, A-549, Hep-2, 293, and human foreskin fibroblast cells; (ii) negative immunofluorescent assays for paramyxoviruses (hRSV, parainfluenza viruses 1 to 3) and other respiratory viruses (adenoviruses, influenza viruses A and B); and (iii) no hemadsorption of human red blood cells. In addition, 10 nasopharyngeal aspirates (NPA) collected during the winter of 2002 as part of a prospective surveillance study in hospitalized children and shown to be PCR positive for the hMPV N gene were included in this study.#}^-g2], 百拇医药
RNA extraction and cDNA synthesis. Total RNA was extracted from 200 µl of infected cell-culture supernatants or NPA specimens using the QIAamp viral RNA minikit (Qiagen, Mississauga, Ontario, Canada). Complementary cDNA was synthesized using 10 µl of eluted RNA, one of the specific hMPV primer (Table 1), and the Omniscript reverse transcriptase kit (Qiagen) according to the manufacturer's instructions.
fig.ommitted!1, 百拇医药
TABLE 1. Sequences of primers used for amplification of hMPV!1, 百拇医药
Primer selection and PCR assays for hMPV. Five different pairs of hMPV primers located in the N, M, F, P, and L genes (Table 1) were selected based on sequence homology with the prototype strain from The Netherlands (GenBank accession number af371337). cDNA was amplified using a real-time PCR procedure with the LC Faststart DNA Master SYBR Green 1 Kit (Roche Diagnostics, Laval, Quebec, Canada) in a LightCycler instrument (Roche Diagnostics). Each reaction had a total volume of 20 µl including 2 µl of cDNA and 18 µl of a reaction mixture containing 4 mM MgCl2, 2 µl of Faststart DNA SYBR Green 1 Master Mix, 3% dimethyl sulfoxide, and a 0.5 µM concentration of each hMPV primer (Table 1). Cycling conditions typically included an initial denaturation step of 10 min at 94°C, followed by 50 cycles of 15 s at 94°C, 5 s at 54°C, and 30 s at 72°C. However, the annealing temperature could slightly vary according to the hMPV gene to amplify. The detection of hMPV amplicons was verified using the melting curve analysis feature of the LightCycler instrument. Briefly, following the last amplification cycle, the internal temperature of the LightCycler was rapidly increased to 94°C then decreased to 60°C for 30 s, followed by a slow increase to 94°C at a rate of 0.1°C per s, with continuous fluorescence reading. The specific melting temperature (Tm) was recorded for each amplified hMPV gene.
DNA sequencing of the hMPV F gene. Nucleotide sequences were determined by automated DNA sequencing (ABI Prism 3100; Applied Biosystems, Foster City, Calif.) using amplified hMPV F gene products purified with the QIAquick PCR purification kit (QIAGEN). Sequences were aligned with the prototype hMPV strain from The Netherlands (af371337) and with previously reported Canadian sequences (2). A phylogenetic tree was constructed using the neighbor joining algorithm and Kimura-2 parameters.-/6v, 百拇医药
RESULTS-/6v, 百拇医药
Comparison of different primer sets for hMPV amplification. A total of 20 LLC-MK2-infected cell cultures with characteristics compatible with hMPV cytopathic effect (2) were selected from the Québec City Virology Laboratory between the years 2000 and 2002. An aliquot of 200 µl of infected LLC-MK2 cells and supernatants was used for RNA extraction followed by cDNA synthesis and real-time PCR amplification as described in the methods section. The presence of hMPV-amplified fragments was first verified by determination of the specific Tm of the amplicons with the LightCycler instrument followed by confirmation of the predicted size of the PCR product by standard ethidium bromide-stained agarose gel electrophoresis and sequence identification of the amplicons (F and N genes only). As shown in Table 2, the real-time PCR assay using primers complementary to the N gene successfully amplified viral RNA from all 20 putative hMPV cultures whereas the PCR positivity rate was somewhat lower for the other genes, i.e., 15 of 20 (75%), 11 of 20 (55%), 12 of 20 (60%), and 18 of 20 (90%) with the M, F, P, and L primers, respectively.
fig.ommitted/n^2q|&, 百拇医药
TABLE 2. Comparative evaluation of different primers for detection of hMPV/n^2q|&, 百拇医药
The different hMPV primers were also evaluated for their ability to detect hMPV RNA in 10 NPA samples collected in a pediatric surveillance study during the winter of 2002 and found to be positive for the hMPV N gene by real-time PCR. In that evaluation, the PCR positivity rate was 6 of 10 (60%), 8 of 10 (80%), 3 of 10 (30%), and 8 of 9 (88.9%), using primers for the M, F, P, and L gene, respectively (Table 2). Overall, sensitivity values of the real-time PCR assays for the hMPV M, F, P, and L genes were 70.0, 63.3, 50.0, and 89.6% when compared to that of the N gene for both viral isolates and clinical samples./n^2q|&, 百拇医药
Determination of hMPV F genotypes. Twelve PCR products were sequenced and aligned to determine their specific F genotype (2, 14). Nine (75.0%) of the 12 sequences were found to cluster with the F-1 genotype which includes the prototype hMPV strain af371337 from which all primer sequences originated whereas the other 3 (25.0%) belonged to the F-2 genotype.
Evaluation of the hMPV N PCR assay. Due to the superior sensitivity of the N primers for amplification of hMPV RNA from both viral isolates and clinical samples, a thorough evaluation of this real-time PCR assay was undertaken. An hMPV plasmid was constructed by subcloning the amplified hMPV N region in the pDrive plasmid (QIAGEN). The new plasmid was transcribed using the RNA Transcription kit (Stratagene, Vancouver, British Columbia, Canada) for sensitivity analysis. Using the previously described RT procedure (N-1 primer) and real-time PCR protocol (N-2 and N-3 primers) (Table 1), the analytical sensitivity of the assay was estimated at 100 copies per reaction. The mean Tm values obtained for the different PCR products amplified from 20 viral cultures and 10 NPA samples were, respectively, 82.70 ± 0.43°C and 82.18 ± 0.33°C, with an overall value of 82.63 ± 0.87°C (Fig. 1). The assay was found to be specific with no amplification signal observed when viral cultures positive for hRSV, parainfluenza viruses 1 to 3, influenza viruses A and B, rhinoviruses, and adenoviruses were tested. A linear amplification was observed over a wide range (>4 log 10) when serial dilutions of the hMPV N plasmid were tested (Fig. 2).
fig.ommitted3$.?cgj, http://www.100md.com
FIG. 1. Melting curve (Tm) analysis of amplified human metapneumovirus strains using primers specific for the nucleoprotein (N) gene.3$.?cgj, http://www.100md.com
fig.ommitted3$.?cgj, http://www.100md.com
FIG. 2. Amplification plots showing threshold cycle (CT) values obtained for serial dilutions (50 to 1,000,000 copies) of a plasmid containing the human metapneumovirus nucleoprotein (N) gene.3$.?cgj, http://www.100md.com
DISCUSSION3$.?cgj, http://www.100md.com
Many viruses including influenza viruses, hRSV, parainfluenza viruses, adenoviruses, rhinoviruses, and coronaviruses have been associated with acute respiratory tract infections (ARTI) of children and adults. Recent investigations have shown that hMPV can now be added to the list of human respiratory pathogens in all age groups (2, 12). In this study, we performed the first systematic evaluation of RT-PCR assays for detection of this new viral pathogen using both infected cell cultures and respiratory specimens. Our results indicate that PCR assays designed to amplify the N and L genes of hMPV have the greatest diagnostic potential probably because they target more conserved regions of the genome. In addition, we described a sensitive real-time PCR procedure for the N gene allowing rapid amplification and detection of hMPV sequences directly from clinical samples in < 2 h.
HMPV was first isolated in 2001 from nasopharyngeal aspirates recovered from Dutch children (14). However, serological studies indicate that this virus has been circulating in humans for at least 50 years (14). It is postulated that this virus has remained unidentified for many years due to its restricted cell line permissivity and its slow replication kinetics (2, 14). Following the publication of the first complete sequence of one hMPV strain (13), viral genomic fragments have been successfully amplified in respiratory samples of individuals from various parts of the world (2, 4, 12, 14; Nissen et al., letter). These studies have shown that hMPV could be associated with severe lower respiratory tract infections (i.e., bronchiolitis and pneumonitis) in young children, elderly subjects, and immunocompromised patients accounting for 1.5 to 10% of unexplained ARTI in pediatric patients.+t7ay7d, 百拇医药
hMPV is most closely related to the avian pneumovirus, also known as Turkey rhinotracheitis virus, which belongs to the Metapneumovirus genus (16). The metapneumoviruses lack nonstructural proteins and the gene order is different from that of pneumoviruses. Furthermore, the amino acid sequence identity between hMPV (a metapneumovirus) and hRSV (a pneumovirus) is <50% for most homologous proteins (13). Most hMPV and hRSV PCR protocols reported to date have relied on amplification of the L, F, N, or M genes (1-6, 7, 8, 11, 12, 15) although no systematic evaluation of different primer sets has been performed. Our results indicate that primers designed to amplify the N and L genes, two internal hMPV genes, are best suitable for diagnostic purposes. The lower PCR positivity rates for the other hMPV genes, i.e., M, F, and P, may be due to the selection of less efficient primer sets, less optimal PCR conditions or the presence of mismatch sequences at the primer hybridization sites. In this regard, phylogenetic analyses have revealed significant sequence variation and the existence of at least two potential genetic clusters of hMPV isolates mainly based on F gene sequences but also on limited sequence information for some of the other genes (2, 10, 14). Since all primer sequences were derived from the prototype strain 001 (GenBank accession number af371337) from The Netherlands which belongs to the putative genotype 1, our PCR protocols may have been suboptimal for the detection of strains belonging to the other genotype(s). Indeed, 9 (75%) of the 12 amplified F gene products that were sequenced in that study clustered with strain 001 in phylogenetic studies (data not shown). However, all culture isolates predicted to contain hMPV and tested in this study were positive by RT-PCR for the N gene which indicates that primers selected in that gene target conserved hMPV sequences.
The real-time RT-PCR for the hMPV N gene offers many advantages over other conventional methods (cell culture) or previously reported RT-PCR protocols. First, this RT-PCR assay is sufficiently sensitive (limit of detection of 100 copies per reaction) for use with clinical samples avoiding lengthy and complicated nested PCR protocols. Also, the continuous monitoring of amplicon development in the LightCycler instrument allows a rapid turnaround time and prevents amplicon contamination. Identification of hMPV-specific products is easily confirmed by a simple determination of the amplicon's Tm value with the LightCycler software. The Tm value obtained for the hMPV N products was found to be very stable and independent of the hMPV genotype (Table 2 and Fig. 1). Alternatively, a TaqMan oligoprobe (5) or adjacent fluorescent hybridization probes can be used with our real-time PCR protocol to obtain more reliable quantitative results.l, http://www.100md.com
In summary, RT-PCR is currently the method of choice for hMPV diagnostic due to the slow replication of this virus in very few cell lines and the unavailability of rapid antigenic detection methods. By comparing PCR protocols for 5 of the 8 hMPV genes, we showed that the N and L genes are probably the best diagnostic targets. Nevertheless, the availability of additional hMPV sequences from various parts of the world is required to confirm these findings. Importantly, the use of rapid and sensitive hMPV PCR assays such as the ones described here should improve our knowledge on the clinical role of this newly described viral pathogen.
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Abels, S., D. Nadal, A. Stroehle, and W. Bossart. 2001. Reliable detection of respiratory syncytial virus infection in children for adequate hospital infection control management. J. Clin. Microbiol. 39:3135-3139.65#h!, http://www.100md.com
Boivin, G., Y. Abed, G. Pelletier, L. Ruel, D. Moisan, S. Côté, T. Peret, D. Erdman, and L. Anderson. 2002. Virological features and clinical manifestations associated with the human metapneumovirus, a new paramyxovirus responsible for acute respiratory tract infections in all age groups. J. Infect. Dis. 186:1330-1334.65#h!, http://www.100md.com
Falsey, A. R., M. A. Formica, and E. E. Walsh. 2002. Diagnosis of respiratory syncytial virus infection: comparison of reverse transcription-PCR to viral culture and serology in adults with respiratory illness. J. Clin. Microbiol. 40:817-820.65#h!, http://www.100md.com
Jartti, T., B. van den Hoogen, R. P. Garofalo, A. D. Osterhaus, and O. Ruuskanen. 2002. Metapneumovirus and acute wheezing in children. Lancet 360:1393-1394.65#h!, http://www.100md.com
Mackay, I. M., K. C. Jacob, D. Woolhouse, K. Waller, M. W. Syrmis, D. M. Whiley, D. J. Siebert, M. Nissen, and T. P. Sloots. 2003. Molecular assays for detection of human metapneumovirus. J. Clin. Microbiol. 41:100-105.
Mazzulli, T., T. C. Peret, A. McGeer, D. Cann, K. S. MacDonald, R. Chua, D. D. Erdman, and L. J. Anderson. 1999. Molecular characterization of a nosocomial outbreak of human respiratory syncytial virus on an adult leukemia/lymphoma ward. J. Infect. Dis. 180:1686-1689.p#v;|f%, http://www.100md.com
Ong, G. M., D. E. Wyatt, H. J. O'Neill, C. McCaughey, and P. V. Coyle. 2001. A comparison of nested polymerase chain reaction and immunofluorescence for the diagnosis of respiratory infections in children with bronchiolitis, and the implications for a cohorting strategy. J. Hosp. Infect. 49:122-128.p#v;|f%, http://www.100md.com
Osiowy, C. 1998. Direct detection of respiratory syncytial virus, parainfluenza virus, and adenovirus in clinical respiratory specimens by a multiplex reverse transcription-PCR assay. J. Clin. Microbiol. 36:3149-3154.p#v;|f%, http://www.100md.com
Pelletier, G., P. Dery, Y. Abed, and G. Boivin. 2002. Respiratory tract reinfections by the new human metapneumovirus in an immunocompromised child. Emerg. Infect. Dis. 8:976-978.p#v;|f%, http://www.100md.com
Peret, T. C., G. Boivin, Y. Li, M. Couillard, C. Humphrey, A. D. Osterhaus, D. D. Erdman, and L. J. Anderson. 2002. Characterization of human metapneumoviruses isolated from patients in North America. J. Infect. Dis. 185:1660-1663.
Stockton, J., J. S. Ellis, M. Saville, J. P. Clewley, and M. C. Zambon. 1998. Multiplex PCR for typing and subtyping influenza and respiratory syncytial viruses. J. Clin. Microbiol. 36:2990-2995.8s)9'6y, 百拇医药
Stockton, J., I. Stephenson, D. Fleming, and M. Zambon. 2002. Human metapneumovirus as a cause of community-acquired respiratory illness. Emerg. Infect. Dis. 8:897-901.8s)9'6y, 百拇医药
van den Hoogen, B. G., T. M. Bestebroer, A. D. Osterhaus, and R. A. Fouchier. 2002. Analysis of the genomic sequence of a human metapneumovirus. Virology 295:119-132.8s)9'6y, 百拇医药
van den Hoogen, B. G., J. C. de Jong, J. Groen, T. Kuiken, R. de Groot, R. A. Fouchier, and A. D. Osterhaus. 2001. A newly discovered human pneumovirus isolated from young children with respiratory tract disease. Nat. Med. 7:719-724.8s)9'6y, 百拇医药
Whiley, D. M., M. W. Syrmis, I. M. Mackay, and T. P. Sloots. 2002. Detection of human respiratory syncytial virus in respiratory samples by LightCycler reverse transcriptase PCR. J. Clin. Microbiol. 40:4418-4422.8s)9'6y, 百拇医药
Yu, Q., P. J. Davis, J. Li, and D. Cavanagh. 1992. Cloning and sequencing of the matrix protein (M) gene of turkey rhinotracheitis virus reveal a gene order different from that of respiratory syncytial virus. Virology 186:426-434.(Stéphanie Côté Yacine Abed and Guy Boivin)
Received 16 January 2003/ Returned for modification 13 March 2003/ Accepted 5 May 2003:, http://www.100md.com
ABSTRACT:, http://www.100md.com
The human metapneumovirus (hMPV) is a new member of the Paramyxoviridae family associated with acute respiratory tract infections in humans. The objective of this study was to compare the sensitivity of real-time RT-PCR assays performed in a LightCycler instrument and designed to amplify the viral nucleoprotein (N), matrix (M), fusion (F), phosphoprotein (P), and polymerase (L) genes. In a first evaluation of 20 viral cultures with characteristics compatible with hMPV cytopathic effect, the PCR positivity rates were 100, 90, 75, 60, and 55% using primers for the N, L, M, P, and F genes. In a second evaluation of 10 nasopharyngeal aspirates from children with bronchiolitis and found to be positive for the hMPV N gene, the PCR positivity rates for the L, M, P, and F genes were 90, 60, 30, and 80%, respectively. The analytic sensitivity of the real-time RT-PCR assay for the hMPV N gene was 100 copies using a transcribed viral plasmid. In conclusion, real-time PCR assays aimed at amplifying the N and L genes which are coding for two internal viral proteins appear particularly suitable for hMPV diagnostic.
INTRODUCTION9m11-v2, 百拇医药
The human metapneumovirus (hMPV) is a newly identified negative-sense RNA virus that has been preliminary assigned to the Metapneumovirus genus within the Paramyxoviridae family (13, 14). This virus differs from the human respiratory syncytial virus (hRSV), another paramyxovirus belonging to the Pneumovirus genus, by virtue of a lack of two nonstructural proteins (NS1 and NS2) and by a different genomic organization for the other common genes, i.e., the nucleoprotein (N), phosphoprotein (P), matrix (M), fusion (F), polymerase (L), attachment glycoprotein (G), M2, and small hydrophobic protein (SH).9m11-v2, 百拇医药
HMPV has been shown to replicate slowly in a few cell lines including tertiary monkey kidney (TMK) cells with cytopathic effects consisting mainly of syncytium formation appearing more than 10 to 14 days postinoculation (2, 14). Virus-infected TMK cell-culture supernatants also do not display any hemagglutinating activity. Molecular detection of hMPV in clinical samples has been mainly achieved by amplifying the F, N, or L genes although no systematic evaluation of such different PCR assays has been performed (2, 5, 12, 14; M. D. Nissen, D. J. Siebert, I. M. Mackay, T. P. Sloots, and S. J. Withers, Letter, Med. J. Aust. 176:188, 2002). A rapid and sensitive PCR assay for hMPV is required in view of its relatively inefficient growth using conventional cell culture methods and its role as a respiratory pathogen in young children, elderly subjects, and immunocompromised patients (2, 4, 9, 14; Nissen et al., letter). In this study, we sought to compare various real-time PCR assays for optimal detection of hMPV in both infected cell cultures and respiratory clinical specimens.
MATERIALS AND METHODS#}^-g2], 百拇医药
Viral isolates and clinical samples. A subset of 20 viral isolates were selected from respiratory samples collected between 2000 and 2002 on the basis of the following characteristics: (i) slow growth on LLC-MK2 cells (a continuous cell line from rhesus monkey kidneys) with no growth on Madin-Darby canine kidney, Vero, A-549, Hep-2, 293, and human foreskin fibroblast cells; (ii) negative immunofluorescent assays for paramyxoviruses (hRSV, parainfluenza viruses 1 to 3) and other respiratory viruses (adenoviruses, influenza viruses A and B); and (iii) no hemadsorption of human red blood cells. In addition, 10 nasopharyngeal aspirates (NPA) collected during the winter of 2002 as part of a prospective surveillance study in hospitalized children and shown to be PCR positive for the hMPV N gene were included in this study.#}^-g2], 百拇医药
RNA extraction and cDNA synthesis. Total RNA was extracted from 200 µl of infected cell-culture supernatants or NPA specimens using the QIAamp viral RNA minikit (Qiagen, Mississauga, Ontario, Canada). Complementary cDNA was synthesized using 10 µl of eluted RNA, one of the specific hMPV primer (Table 1), and the Omniscript reverse transcriptase kit (Qiagen) according to the manufacturer's instructions.
fig.ommitted!1, 百拇医药
TABLE 1. Sequences of primers used for amplification of hMPV!1, 百拇医药
Primer selection and PCR assays for hMPV. Five different pairs of hMPV primers located in the N, M, F, P, and L genes (Table 1) were selected based on sequence homology with the prototype strain from The Netherlands (GenBank accession number af371337). cDNA was amplified using a real-time PCR procedure with the LC Faststart DNA Master SYBR Green 1 Kit (Roche Diagnostics, Laval, Quebec, Canada) in a LightCycler instrument (Roche Diagnostics). Each reaction had a total volume of 20 µl including 2 µl of cDNA and 18 µl of a reaction mixture containing 4 mM MgCl2, 2 µl of Faststart DNA SYBR Green 1 Master Mix, 3% dimethyl sulfoxide, and a 0.5 µM concentration of each hMPV primer (Table 1). Cycling conditions typically included an initial denaturation step of 10 min at 94°C, followed by 50 cycles of 15 s at 94°C, 5 s at 54°C, and 30 s at 72°C. However, the annealing temperature could slightly vary according to the hMPV gene to amplify. The detection of hMPV amplicons was verified using the melting curve analysis feature of the LightCycler instrument. Briefly, following the last amplification cycle, the internal temperature of the LightCycler was rapidly increased to 94°C then decreased to 60°C for 30 s, followed by a slow increase to 94°C at a rate of 0.1°C per s, with continuous fluorescence reading. The specific melting temperature (Tm) was recorded for each amplified hMPV gene.
DNA sequencing of the hMPV F gene. Nucleotide sequences were determined by automated DNA sequencing (ABI Prism 3100; Applied Biosystems, Foster City, Calif.) using amplified hMPV F gene products purified with the QIAquick PCR purification kit (QIAGEN). Sequences were aligned with the prototype hMPV strain from The Netherlands (af371337) and with previously reported Canadian sequences (2). A phylogenetic tree was constructed using the neighbor joining algorithm and Kimura-2 parameters.-/6v, 百拇医药
RESULTS-/6v, 百拇医药
Comparison of different primer sets for hMPV amplification. A total of 20 LLC-MK2-infected cell cultures with characteristics compatible with hMPV cytopathic effect (2) were selected from the Québec City Virology Laboratory between the years 2000 and 2002. An aliquot of 200 µl of infected LLC-MK2 cells and supernatants was used for RNA extraction followed by cDNA synthesis and real-time PCR amplification as described in the methods section. The presence of hMPV-amplified fragments was first verified by determination of the specific Tm of the amplicons with the LightCycler instrument followed by confirmation of the predicted size of the PCR product by standard ethidium bromide-stained agarose gel electrophoresis and sequence identification of the amplicons (F and N genes only). As shown in Table 2, the real-time PCR assay using primers complementary to the N gene successfully amplified viral RNA from all 20 putative hMPV cultures whereas the PCR positivity rate was somewhat lower for the other genes, i.e., 15 of 20 (75%), 11 of 20 (55%), 12 of 20 (60%), and 18 of 20 (90%) with the M, F, P, and L primers, respectively.
fig.ommitted/n^2q|&, 百拇医药
TABLE 2. Comparative evaluation of different primers for detection of hMPV/n^2q|&, 百拇医药
The different hMPV primers were also evaluated for their ability to detect hMPV RNA in 10 NPA samples collected in a pediatric surveillance study during the winter of 2002 and found to be positive for the hMPV N gene by real-time PCR. In that evaluation, the PCR positivity rate was 6 of 10 (60%), 8 of 10 (80%), 3 of 10 (30%), and 8 of 9 (88.9%), using primers for the M, F, P, and L gene, respectively (Table 2). Overall, sensitivity values of the real-time PCR assays for the hMPV M, F, P, and L genes were 70.0, 63.3, 50.0, and 89.6% when compared to that of the N gene for both viral isolates and clinical samples./n^2q|&, 百拇医药
Determination of hMPV F genotypes. Twelve PCR products were sequenced and aligned to determine their specific F genotype (2, 14). Nine (75.0%) of the 12 sequences were found to cluster with the F-1 genotype which includes the prototype hMPV strain af371337 from which all primer sequences originated whereas the other 3 (25.0%) belonged to the F-2 genotype.
Evaluation of the hMPV N PCR assay. Due to the superior sensitivity of the N primers for amplification of hMPV RNA from both viral isolates and clinical samples, a thorough evaluation of this real-time PCR assay was undertaken. An hMPV plasmid was constructed by subcloning the amplified hMPV N region in the pDrive plasmid (QIAGEN). The new plasmid was transcribed using the RNA Transcription kit (Stratagene, Vancouver, British Columbia, Canada) for sensitivity analysis. Using the previously described RT procedure (N-1 primer) and real-time PCR protocol (N-2 and N-3 primers) (Table 1), the analytical sensitivity of the assay was estimated at 100 copies per reaction. The mean Tm values obtained for the different PCR products amplified from 20 viral cultures and 10 NPA samples were, respectively, 82.70 ± 0.43°C and 82.18 ± 0.33°C, with an overall value of 82.63 ± 0.87°C (Fig. 1). The assay was found to be specific with no amplification signal observed when viral cultures positive for hRSV, parainfluenza viruses 1 to 3, influenza viruses A and B, rhinoviruses, and adenoviruses were tested. A linear amplification was observed over a wide range (>4 log 10) when serial dilutions of the hMPV N plasmid were tested (Fig. 2).
fig.ommitted3$.?cgj, http://www.100md.com
FIG. 1. Melting curve (Tm) analysis of amplified human metapneumovirus strains using primers specific for the nucleoprotein (N) gene.3$.?cgj, http://www.100md.com
fig.ommitted3$.?cgj, http://www.100md.com
FIG. 2. Amplification plots showing threshold cycle (CT) values obtained for serial dilutions (50 to 1,000,000 copies) of a plasmid containing the human metapneumovirus nucleoprotein (N) gene.3$.?cgj, http://www.100md.com
DISCUSSION3$.?cgj, http://www.100md.com
Many viruses including influenza viruses, hRSV, parainfluenza viruses, adenoviruses, rhinoviruses, and coronaviruses have been associated with acute respiratory tract infections (ARTI) of children and adults. Recent investigations have shown that hMPV can now be added to the list of human respiratory pathogens in all age groups (2, 12). In this study, we performed the first systematic evaluation of RT-PCR assays for detection of this new viral pathogen using both infected cell cultures and respiratory specimens. Our results indicate that PCR assays designed to amplify the N and L genes of hMPV have the greatest diagnostic potential probably because they target more conserved regions of the genome. In addition, we described a sensitive real-time PCR procedure for the N gene allowing rapid amplification and detection of hMPV sequences directly from clinical samples in < 2 h.
HMPV was first isolated in 2001 from nasopharyngeal aspirates recovered from Dutch children (14). However, serological studies indicate that this virus has been circulating in humans for at least 50 years (14). It is postulated that this virus has remained unidentified for many years due to its restricted cell line permissivity and its slow replication kinetics (2, 14). Following the publication of the first complete sequence of one hMPV strain (13), viral genomic fragments have been successfully amplified in respiratory samples of individuals from various parts of the world (2, 4, 12, 14; Nissen et al., letter). These studies have shown that hMPV could be associated with severe lower respiratory tract infections (i.e., bronchiolitis and pneumonitis) in young children, elderly subjects, and immunocompromised patients accounting for 1.5 to 10% of unexplained ARTI in pediatric patients.+t7ay7d, 百拇医药
hMPV is most closely related to the avian pneumovirus, also known as Turkey rhinotracheitis virus, which belongs to the Metapneumovirus genus (16). The metapneumoviruses lack nonstructural proteins and the gene order is different from that of pneumoviruses. Furthermore, the amino acid sequence identity between hMPV (a metapneumovirus) and hRSV (a pneumovirus) is <50% for most homologous proteins (13). Most hMPV and hRSV PCR protocols reported to date have relied on amplification of the L, F, N, or M genes (1-6, 7, 8, 11, 12, 15) although no systematic evaluation of different primer sets has been performed. Our results indicate that primers designed to amplify the N and L genes, two internal hMPV genes, are best suitable for diagnostic purposes. The lower PCR positivity rates for the other hMPV genes, i.e., M, F, and P, may be due to the selection of less efficient primer sets, less optimal PCR conditions or the presence of mismatch sequences at the primer hybridization sites. In this regard, phylogenetic analyses have revealed significant sequence variation and the existence of at least two potential genetic clusters of hMPV isolates mainly based on F gene sequences but also on limited sequence information for some of the other genes (2, 10, 14). Since all primer sequences were derived from the prototype strain 001 (GenBank accession number af371337) from The Netherlands which belongs to the putative genotype 1, our PCR protocols may have been suboptimal for the detection of strains belonging to the other genotype(s). Indeed, 9 (75%) of the 12 amplified F gene products that were sequenced in that study clustered with strain 001 in phylogenetic studies (data not shown). However, all culture isolates predicted to contain hMPV and tested in this study were positive by RT-PCR for the N gene which indicates that primers selected in that gene target conserved hMPV sequences.
The real-time RT-PCR for the hMPV N gene offers many advantages over other conventional methods (cell culture) or previously reported RT-PCR protocols. First, this RT-PCR assay is sufficiently sensitive (limit of detection of 100 copies per reaction) for use with clinical samples avoiding lengthy and complicated nested PCR protocols. Also, the continuous monitoring of amplicon development in the LightCycler instrument allows a rapid turnaround time and prevents amplicon contamination. Identification of hMPV-specific products is easily confirmed by a simple determination of the amplicon's Tm value with the LightCycler software. The Tm value obtained for the hMPV N products was found to be very stable and independent of the hMPV genotype (Table 2 and Fig. 1). Alternatively, a TaqMan oligoprobe (5) or adjacent fluorescent hybridization probes can be used with our real-time PCR protocol to obtain more reliable quantitative results.l, http://www.100md.com
In summary, RT-PCR is currently the method of choice for hMPV diagnostic due to the slow replication of this virus in very few cell lines and the unavailability of rapid antigenic detection methods. By comparing PCR protocols for 5 of the 8 hMPV genes, we showed that the N and L genes are probably the best diagnostic targets. Nevertheless, the availability of additional hMPV sequences from various parts of the world is required to confirm these findings. Importantly, the use of rapid and sensitive hMPV PCR assays such as the ones described here should improve our knowledge on the clinical role of this newly described viral pathogen.
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